Tension sensors reveal how the kinetochore shares its load

Abstract

At metaphase in mitotic cells, pulling forces at the kinetochore-microtubule interface create tension by stretching the centromeric chromatin between oppositely oriented sister kinetochores. This tension is important for stabilizing the end-on kinetochore microtubule attachment required for proper bi-orientation of sister chromosomes as well as for satisfaction of the Spindle Assembly Checkpoint and entry into anaphase. How force is coupled by proteins to kinetochore microtubules and resisted by centromere stretch is becoming better understood as many of the proteins involved have been identified. Recent application of genetically encoded fluorescent tension sensors within the mechanical linkage between the centromere and kinetochore microtubules are beginning to reveal - from live cell assays - protein specific contributions that are functionally important.

Keywords: centromeric chromatin; chromosome segregation; kinetochore; microtubules; mitosis; tension sensor.

Figure 1

Structural organization of the major kinetochore constituents at the microtubule plus-end. 3-D visualization of the metaphase budding yeast kinetochore-microtubule attachment as predicted by the protein localization data assuming a symmetric arrangement of kinetochore protein complexes around the cylindrical microtubule lattice. Ndc80c shown as blue/yellow and red/green coiled-coils in A and B with more detail. A: The MIND complex (Mtw1, Nnf1, Nsl1 and Dsn1) (gray/green ovoids and yellow/blue balls) provide the linkage from Ndc80c to COMA. COMA (Ctf19, Okp1, Mcm21, and Ame1) dk and lt blue triangles and purple spheres. Mif2 is depicted in red, the Cse4 containing nucleosome in shades of purple wrapped by a DNA double strand (yellow fiber). B: Structural view of Ndc80c, including the Ndc80 loop domain and the site of insertion of the FRET biosensor [53].

Figure 2

Three different types of kinetochore microtubule and centromere stretch dynamics in metaphase (M) and anaphase A (A) exhibited by different kinds of mitotic or meiotic eukaryotic cells. Type I: No flux of kinetochore microtubules that extend between the kinetochore and the pole; kMTs lengthen and shorten at their kinetochore-attached plus ends (kinetochore directional instability), e.g. budding yeast. Type II: Flux of kinetochore microtubules and kinetochore directional instability, e.g. animal tissue cells. Type III: Flux of kinetochore microtubules coupled to polymerization at kinetochores until loss of tension in anaphase causes a switch to depolymerization or polymerization at kinetochores much slower than flux [42]); kinetochore oscillations are minimized, e.g. spermatocytes, oocytes, early embryos, and higher plants, Xenopus extract spindles, and Drosophila S2 tissue cells. The kinetochores (yellow box) are relatively stiff in metaphase.

Figure 3

Estimate mean force measured by FRET BioSensor. A: Measured values of FRET efficiency for control metaphase and late anaphase, and for cells treated with low-dose benomyl (55μM). B: FRET efficiency versus Emission Ratio shows linear dependency. C: FRET efficiency versus distance between fluorophores. Inset shows FRET in cell with the Ndc80 biosensor. The outline of the budded cell (white) and clusters of bi-oriented Ndc80 in metaphase are shown. The four dotted lines in the graph represent the position of No tension in solution (lt. brown), Late Anaphase (yellow), Benomyl 55μm (dk brown), and Metaphase (purple). D: Estimated force extension curve calculated using worm like chain (see WLC equation in text).

Figure 4

Models for the Ndc80 force coupler during polymerization in wild type and consequences of Dam1 mutants in which the kinetochore dislocates from the microtubule plus-end [58]. A: During polymerization, force from centromere stretch pulls the Ndc80 force coupler along kMTs with the microtubule binding domains of Dam1 and Ndc80 under tension. During depolymerization, forces from peeling protofilaments push the Dam1 and Ndc80 complexes along kMTs towards the pole to stretch the centromere; the MTBDs of both Dam1 and Ndc80 are under compression (not shown). C: Mutations in Dam1 that increase friction between Dam1 and the microtubule lattice may prevent kinetochores from keeping up with the ends of polymerizing microtubules, leading to dislocation of the kinetochore from the MT plus-end [70]. Ndc80 resistive tension (green arrow in C) from centromere stretch is reduced in this state with increased Dam1 friction (blue arrow in C). B and D: Snapshots of movies derived from 600 s duration simulations presented in [58], showing kinetics for all 16 sister kinetochore pairs and their centromere stretch. See Supporting Information to view the movies. B: WT, D: dam1 mutant.

Figure 5

Packing of pericentric chromatin loops between sister kinetochores in metaphase as a mechanism to increase stiffness. A: The pericentromeric chromatin from one replicated chromosome is shown. The DNA is depicted as beads on a string chromatin (pink circles). The centromeres (defined at the site of attachment to kMTs) from each sister chromatid are at the distal ends of the primary loop, in contact with a kMT (kMTs are in green). The 16 kMTs in the metaphase spindle emanate from the spindle pole body (red oval). The pericentric chromatin is organized as loops radiating from the primary loops (radial loops), depicted as four loops for each sister strand, a total of eight loops between the sister kinetochores. Cohesin (black rings) hold sister chromatid arms together, and pericentric cohesin links intra-strand loops. Chromosome arms (away from the pericentric region) extend north and south. B: Visualization of pericentric DNA in living cells. Left, schematic of spindle poles (red spheres) connected to a circular chromosomal DNA (small spheres) via kinetochore microtubules (black rods). The DNA will appear as a foci off the spindle axis (top) or as a linear array on the spindle axis (bottom). To the right of the schematic are shown images of live cells. The poles are indicated in red and the lacO DNA in green.